The fluid handling and microbiological properties of a non-antimicrobial Hydrofiber® (NAH) wound dressing have been compared with those of a silver salt-containing Hydrofiber® (SCH). Fluorescent dyes (BacLight™, Live/Dead™ Kit) were added to fresh cultures of two wound pathogens (Pseudomonas aeruginosa and Staphylococcus aureus), and used to visualise their viability. Live bacteria stained green and dead/dying bacteria turned red. When inoculated into samples of the NAH and SCH dressings, the viability of the bacteria could be effectively monitored over time using a rapid form of confocal laser scanning microscopy (RCLSM—Leica® UK). When the NAH dressing was hydrated with stained bacterial culture, its fibres swelled quickly, reducing interstitial spaces between the fibres, resulting in the formation of a cohesive gel. Bacteria became immobilised in the gel, forming characteristic clumps, but remained largely green (viable) for more than 20 h with no apparent increase in numbers. The SCH initially behaved in a similar manner, however, using 3-D data from RCLSM time-lapse sequences P. aeruginosa was observed to turn progressively red (i.e. died) within 1.5–3 h and S. aureus similarly turned red within 5–7 h of contact with the SCH dressing. The ability of both Hydrofiber® dressings to sequester and immobilise potentially pathogenic wound micro-organisms has been demonstrated. Additionally the SCH dressing was shown to kill immobilised bacteria, as a consequence of the ionic silver bactericide. These properties of the Hydrofiber® dressings may contribute to providing an environment that is supportive to wound healing.